CD44 is a glycoprotein located on the cell surface which was originally described as a "lymphocyte homing receptor" and would appear to be implicated in the adhesion of lymphocytes to certain mucosal endothelial cells of veins (Peyer's patch or Peyer-Plaques or Folliculi lymphatici aggregati) or postcapillary veins in the lymph nodes (S. T. Jalkanen et al., Eur. J. Immuol. 16:1195-1202 (1986); R. L. Camp et al., J. Exp. Med. 173:763-766 (1991)). In addition, CD44 glycoprotein is thought to be implicated in the maturation and activation of lymphocytes and has a (co)determining effect on the increased migration capacity of all lymphoblasts (e.g. R. L. Camp et al. (1991) loc. cit; S. Huet et al., J. Immunol. 143:798-801 (1989)) and is believed to play a role as an anchor point for other adhesion molecules (Y. Shimizu et al., J. Immunol. 143:2457-2463 (1989)). However, as of the present, CD44 has not been clearly found to possess all these functions.
Recently, it was found, in experiments on rat tumor cells which metastasize via the lymphatic system (BSp73 cells of a spontaneous rat pancreas adenocarcinoma) that these cells express variants of CD44 (vCD44) and are responsible for the "trafficking" of tumor cells. This situation has also been demonstrated on other tumor cell lines.
It has been demonstrated that this vCD44 glycoprotein imparts metastasizing qualities to a tumor which, a prior, does not metastasize, whereas the standard type CD44 (sCD44) is not capable of this. Thus, it can now be assumed that vCD44 as opposed to CD44 is a metastasis-specific protein which enables tumors to metastasize through the lymph tracts (U. Gunthert et al., Cell 65:13-24 (1991)).
Further clarification of the vCD44 glycoprotein of the rat up to the final characterization of the DNA and amino acid sequence was achieved by U. Gunthert et al. (1991), loc. cit., by means of the BSp73 rat cell system which consists of two morphologically or phenotypically different syngeneic cell variants: a non-metastasizing variant AS (BSp73AS) and a metastasizing variant ASML (BSp73ASML) (S. Matzku et al., Cancer Research 49:1294-1299 (1989)).
For this purpose, monoclonal antibodies (mAbs) were prepared which recognize the antigenic determinant on the metastasizing variant BSp73ASML.
Cell lines have been obtained both from the primary tumor (subcutaneous non-metastasizing node consisting of BSp73AS cells) and also a metastasis thereof (BSp73ASML cells which metastasize in lymph nodes and lungs). mAbs were prepared which were directed against the membrane proteins of BSp73ASML cells (S. Matzku et al. (1989), loc. cit.). One of these mAbs, which recognizes only epitopes on BSp73ASML, but not those on BSp73AS cells or other non-tumorigenic cells, was used to search through an E. coli cDNA expression library, prepared from poly(A).sup.+ RNA from BSp73ASML cells and a suitable vector system (screening). In this way it was possible to identify a clone (pMeta-1) which contains the total cDNA with a length of 3207 bp and which codes for an additional domain of 162 amino acids. This domain cannot be found either in sCD44 cells or in other non-metastasizing tumor cells and contains the mAb specific epitope-coding region. Using mRNA preparations from cells from various tissues and mRNA:DNA hybridizations carried out therewith, with different DNA samples obtained from the cDNA clones, the object was to establish that vCD44 is a splicing variant of sCD44 and that the expression of the vCD44 RNAs is closely linked to the formation of metastases. Thus, it is found that the additional extracellular domain coded by the 486 bp long insert (amino acids 224 to 385 in pMeta-1) is the part of the surface glycoprotein vCD44 which is implicated in metastasis.
The metastatic tumor growth (adenocarcinoma in the rat) was successfully suppressed after immunization with monoclonal antibodies which recognize the above-mentioned epitope or which specifically react with this extracellular region of vCD44 (S. Reber et al., Int. J. Cancer 46:919-927 (1990)).
The identification of this extracellular variant domain in the rat (pMeta-1 or rMeta-1) also makes it possible to clarify the equivalent human nucleotide and amino acid sequences:
Using a cDNA probe derived from the DNA of the vCD44 domain in the rat homologous regions in these DNAs can be found by hybridization, optionally under stringent conditions, with genomic DNA from various species-specific cell lines (e.g. human, rat, mouse). A probe of this kind can subsequently be used for hybridizing against RNAs from various human cell lines, especially tumor cell lines, e.g. large-cell lung cancers, melanomas, colon carcinomas, breast tumors, keratinocytes. In this way a suitable human cell line, e.g. that of a large-cell lung cancer, can readily be found containing sequences which are homologous to the cDNA probe of the rat. By PCR (polymerase chain reaction), a known in vitro method of selectively concentrating DNA regions of a specific length and specific sequence from a mixture of DNA molecules, using a DNA polymerase and a suitable primer, it is possible to obtain cDNAs from RNA preparations of various human cell lines, e.g. cells of large-cell lung cancers, melanomas, colon carcinomas and immortalized keratinocytes, which code for sCD44 and vCD44.
A cDNA of this kind obtained by PCR can be ligated into a suitable cloning vector by known methods and subsequently sequenced, after corresponding cultivation of the host cells transformed therewith, preferably bacteria such as E. coli. In this way, it is possible to obtain, from a variety of human cells selected from all possible tumor cells or cell lines, especially those which have metastasizing properties, DNA sequences which code either for a normal human sCD44 glycoprotein of a known order of magnitude in the region of 350 amino acids (I. Stamenkovic et al., Cell 56: 1057-1062 (1989)) which contains an extracellular domain in the region of 85 kDa (S. Jalkanen et al., J. Cell Bol. 105:983-990 (1987)) or which code for a variant human vCD44 glycoprotein which may comprise an additional extracellular domain of varying lengths, more particularly between 850 bp and 1.5 kb, for example 1014 bp (or 338 amino acids), and which is inserted in the DNA sequence coding for the normal sCD44 glycoprotein, for example between the positions of the nucleotides 782 and 783 (FIG. 4). A DNA sequence of this kind may occur in several, e.g. five domains which represent different exons and may be found both in various animal cell lines (e.g. the rat or mouse) and also in human cell lines.
The cDNA shown by way of example in FIG. 4A, which is homologous with the longest variant section of human tumor cell lines obtained, was isolated from the rat tumor cell line BSpASML using PCR. A direct comparison of the amino acid sequence derived therefrom with that of the human clone obtained from a human tumor cell line shows that domain I has 83%, domain II has 83%, domain III has 71%, domain IV has 82% and domain V has 66% homology with the rat DNA sequence. Overall this means that about 76% of the human sequences are conserved in the variant regions as compared with the rat sequence. The corresponding rat sequences which impart the metastatic potential to the tumor (U. Gunthert et al. (1991), loc. cit.) comprise amino acids 258 to 420 and are coded by the domains II and III.
It was thus clear to those skilled in the art that poly- or monoclonal antibodies which specifically recognize these epitopes, i.e. this additional extracellular region on various splicing variants of vCD44 and react therewith, and which may be labelled with radioisotopes and/or conjugated with cytocidal or cytotoxic substances, may be used for diagnostic and therapeutic purposes in the treatment of metastasizing tumors in humans (see PCT WO 91/17248).
It has now, surprisingly, been found that antibodies, especially monoclonal antibodies which react with the various metastasis-specific variants of CD44 (vCD44) have an immunosuppressant activity; this new property could not have been foreseen under any circumstances in the light of the facts outlined above.